Wild type p53 stabilization and activation in response to stress is critical for its tumor suppressor function. Paradoxically, mutant p53 is also highly stabilized in tumors and promotes metastasis and therapy resistance through gain-of-function. Elucidating the mechanisms that regulate p53 stability and activation may provide novel therapeutic strategies that target wild type or mutant p53. MDM2 and MDMX are major regulators of p53 ubiquitination, DNA binding, and transcriptional activity. Our recent study suggested that ATM-dependent phosphorylation of MDM2 and MDMX near the RING domains are important for stabilization and activation of p53. We found that these phosphorylation events inhibit the homo-dimerization of MDM2 RING domain, inhibit the intra- molecular interaction between the MDM2 RING and acidic domains, and inhibit the intra-molecular interaction between MDMX acidic and p53 binding domains. We also identified the MDM2 acidic domain as an important activator of the RING domain E3 ligase activity. These findings led to the hypothesis that various signaling pathways target the intra-molecular interactions in MDM2 and MDMX to stabilize and activate p53. The following experiments are proposed to further study the mechanisms of p53 activation during stress response. (1) Determine the function and mechanism of the MDM2 acidic domain. (2) Investigate the regulation of MDM2 domain interactions by stress signals. (3) Test a novel strategy of targeting intra-molecular interactions (4) Characterize the structure and dynamics of the MDM2 and MDMX domain interactions. The proposed experiments will lead to a new level of understanding of p53 regulation, explore novel intra- molecular regulatory mechanisms of MDM2 and MDMX, and develop new strategies for investigating and targeting MDM2 and MDMX.